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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
FromApril 20,2012 to June 14,2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP complint with international guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment no. 113 - Supplement to Test Guidelines 437 and 438: Collection of Tissues for Histopathological Evaluation and Collection of Data on Non severe Irritants.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1
- Substance type:organic
- Physical state:Amber-yellowish brown undefined liquid
- Appearance at first use: Ivory dense cream
- Storage: Ambient condition

Test animals / tissue source

Species:
other: Bovine eye
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: Butcher Service s.r.l. - Mattatoio no. 2067 M
- Age of animals: 6-12 months
- Killing time: From 9:30 to 11:30 in the morning.
- Transport condition: Maintained in eyes transport solution at approximately 4°C.
- Storage condition: These will be placed into plastic containers with approximately 1 litre of Hanks balanced salt solution (HBSS) for every 25 eyes collected. The time of killing of the animals will be estimated. The containers will be transported to the test facility in refrigerated condition (approximately 4°C) and the time of arrival will be recorded.
- Eye health: all eyes will be carefully examined. Any defects such as opacity, scratches or pitting of the corneal surface, vascularisation or pigmentation will render the eye unsuitable for use

Test system

Vehicle:
other: EMEM
Controls:
other: not required
Amount / concentration applied:
Fresh solutions/suspensions of the test item will be prepared for each day's work; solutions/suspensions will be prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test item.
- Liquid test items: with the exception of surfactants, will be used as supplied.
- Solid substances: with the exception of surfactants, will be dissolved/suspended to 20% (w/v) concentration in Eagle’s Minimum Essential Medium (EMEM), sterile deionized water or physiological saline (NaCl 0.9%). Paraffin oil may be used for hydrophobic substances. If necessary, they will be ground to reduce particle size and aid suspension.
- Surfactants will be dissolved/diluted to 10% (v/v or w/v) concentration using EMEM, sterile deionized water or physiological saline.
- Surfactant preparations will be tested neat, unless differently indicated by the Sponsor. Dilution/suspensions using EMEM, sterile deionized water or physiological saline may be evaluated



Duration of treatment / exposure:
Corneas were exposed in horizontal position for 10 ± 1 minutes, incubated in a liquid bath at 32 ± 1°C
Observation period (in vivo):
Corneas were maintained in horizontal position, for further 2 hours ± 10 minutes, incubated in a liquid bath at 32 ± 1°C.
Number of animals or in vitro replicates:
Once underwent the first examination, a total of 10 corneas were processed (out of 12 delivered) for a final selection of the 9 required corneas according to basal opacity.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Time after start of exposure:10 minutes
SCORING SYSTEM:according to OECD 437
- Washing: After exposure, corneas were rinsed thoroughly with complete EMEM with phenol red (3 washing with 3 mL each). A final wash with prewarmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled with prewarmed complete EMEM without phenol red.

TOOL USED TO ASSESS SCORE: fluorescein

other:
CONTROL ITEMS
Positive control item: 1% (w/v) sodium hydroxide (NaOH; Sigma, batch no. BCBG2025V) in water (Baxter, batch no. 11K0903), being the test item a liquid.
Negative control item: Physiological saline (0.9% NaCl; BieffeMedital batch no. 10I0208) as a control of the test system.

Media
Eyes transport solution: Hanks balanced salt solution (HBSS) Modified supplemented with Penicillin sulphate and Streptomycin sulphate both at the final concentration of 100 IU/mL.
Complete EMEM*
- without phenol red: EMEM* Gibco (Invitrogen) Cat. no. 51200 supplemented with 1% (v/v) Foetal Bovine Serum (FBS - Hyclone - batch no. ATJ33161)
- with phenol red: EMEM* Gibco (Invitrogen) Cat. no. 21090 supplemented with 1% (v/v) Foetal Bovine Serum (FBS - Hyclone - batch no. ATJ33161)
* also named: Minimum Essential Medium Eagle's (MEM) Modified or MEM or Eagle’s MEM. These media were prewarmed in a water bath at 32°C during the experimental procedure.
Sodium fluorescein solution: Sigma Cat. no. F-6377. This was formulated to give 4 mg/mL solutions in DPBS [Gibco Cat. No. 14190].

Results and discussion

In vivo

Results
Irritation parameter:
other: IVIS
Basis:
mean
Time point:
other: 10 minuts
Score:
ca. 6.1
Max. score:
100
Reversibility:
other: not applicable
Irritant / corrosive response data:
Details of measurements carried out at the observations are reported in Table 2.
The negative control samples did not induce corrosion as the mean opacity and permeability values were homogeneous and in line for the expected values for this kind of control.
The test item slightly increased the corneal opacity, being the calculated mean value 2.1.
The alteration was not homogeneous in the three replicates since in replicate A1 the response was higher than in the remaining two.
At the macroscopic observation the three corneas showed slight opacity.
With reference to the cornea permeability, this was slightly affected when compared to that of negative control, thus indicating a minimal alteration of corneal barrier. Also in this case alteration was higher in replicate A1.
The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 232.6. Opacity and exfoliation were noted in the three replicates at the end of the 4-hour post-incubation period.
The corneal permeability was also increased. The calculated mean permeability OD490 value was 2.4223.

Any other information on results incl. tables

Analysis of data

For each test point, the mean value of opacity obtained after exposure was calculated. As appropriate, negative control was subtracted from the opacity of the test item or positive control.

The mean value of permeability, expressed as optical density units, was also calculated and corrected against the mean negative control value.

The in vitro irritancy score (IVIS) of the test item was calculated from these values in the following manner:

IVIS = mean opacity score + (15 x mean Permeability OD490 score)

Classification of the test item was performed according to the in vitro irritancy score:

Value Classification

> 55.1 Corrosive or severe irritant

In addition, the test item was evaluated also for the eye irritancy potential according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438:

Value Classification

0 -3 Non eye irritant

3.1 -25 Mild eye irritant

25.1 - 55 Moderate eye irritant

> 55.1 Corrosive or severe irritant

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The potential of the test item, PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1, to be severely irritant or corrosive to the eye, was measured by its ability to induce opacity and increase permeability in isolated bovine corneas.
The IVIS was 6.1. The negative and positive controls gave the expected results. The test is therefore considered as valid. According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye. However, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is indication of mild irritant effect to the eye.
Executive summary:

The potential of the test item, PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on 7 September 2009) and OECD Supplement to Test Guidelines nos. 437 and 438.

The test item was used as supplied (being a cream) on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours.

Positive and negative controls [a 1% (w/v) sodium hydroxide solution in water and physiological saline alone, respectively] were concurrently tested in the same number of replicates.

The mean opacity detected with an opacitometer at the end of the test item exposure period was 2.1, slightly higher than that of the negative control. The alteration was not homogeneous in the three replicates since in one replicate the response was higher than in the remaining two.

After the determination of opacity, the epithelial surface was treated with a 0.4% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability.

Mean OD490 value of the corneas treated with the test item was slightly affected when compared to that of negative control, thus indicating a minimal alteration of corneal barrier. Also in this case alteration was higher in the same individual replicate.

Negative and positive controls gave the expected results.

The results obtained indicate that the test item induces slight changes in cornea opacity but especially in corneal permeability under the reported experimental conditions.

The calculated in vitro irritancy score (IVIS) for the test item is 6.1.

According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye.

However, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is indication of mild irritant effect to the eye.